Apicomplexans are among the most common parasites of humans, accounting for significant mortality and morbidity through diseases like malaria, toxoplasmosis and cryptosporidiosis. As obligate intracellular parasites, they establish intimate interactions with their hosts. Toxoplasma gondii is an extreme example of this adaptation, able to replicate within nearly every cell type in any warm-blooded host. Genome-scale analyses will be needed to systematically explore the genetics of host-parasite interactions. We present preliminary evidence for the use of CRISPR/Cas9 to target all predicted protein-coding genes in the parasite. This advance enables rapid and cost-effective assessment of the relative contribution of each parasite gene to growth under different conditions. Using this platform we can identify genes important for parasite survival in genetically diverse hosts. Coevolution between T. gondii and mice is evident in the variable parasite growth observed in cell lines derived from different mouse strains. We therefore propose to measure the effect of host variation on the parasite genes required for optimal growth in fibroblasts. To maximize the resources available to downstream applications we will focus our study on the founder strains for two recombinant inbred (RI) families, the Collaborative Cross (CC) and the BXD family, which together account for more than 90% of the genetic diversity in laboratory mice. The first aim of this study will extend our CRISPR/Cas9-mediated genome-wide analysis to cell lines derived from the nine inbred mouse strains. This aim will define two sets of parasite genes: those important for parasitism across different hosts, and those that only contribute to growth in certain mouse strains. The second aim will examine the phenotypes associated with disrupting specific parasite genes, leveraging the efficiency of CRISPR/Cas9, automated microscopy, and image analysis. Based on our preliminary work, we have selected 14 genes to study, which strongly contribute to fitness in human fibroblasts, are conserved among apicomplexans, and are predicted to be secreted. Study of these important parasite factors will extend our understanding of host-parasite interactions, and establish the methods for high-throughput phenotyping of parasite genes. These methods will be valuable to investigate the phenotypes of other relevant genes identified in the first aim. The proposed work will be the first systematic analysis of the apicomplexan genome in the context of natural host variation. Defining the categories of genes that broadly or specifically affect infection will helpus understand the cellular basis of parasitism.